Isolation of viable cell types from the gill epithelium of Japanese eel Anguilla japonica.

Abstract

High-purity viable cells with low mitochondria (pavement cells) and mitochondria-rich content (chloride cells) were successfully isolated from the gill epithelium of Japanese eels, using three-step Percoll gradient low-speed centrifugation. Cytochemistry (silver staining for chloride, rhodamine-123, and Mitotracker for mitochondria and actin/spectrin immunofluorescence) and scanning electron microscope images were used to identify the cell types in the gill epithelium of the eel. Pavement cells were isolated at 97 and 98% purity for freshwater- and seawater-adapted eels, respectively, and chloride cells were obtained at 89 and 92% purity. The enzymatic activities of the isolated cells were determined. Na+-K+-ATPase, Mg2+-ATPase, and succinate dehydrogenase were found mainly in the chloride cell. Alkaline Ca2+-ATPase and low- and high-affinity Ca2+-ATPase were about twice as high in the chloride cell compared with the pavement cell. Transfer of eels to seawater resulted in enlargement of chloride cell sizes and significant increases in Na+-K+-ATPase, Mg2+-ATPase, and succinate dehydrogenase activities, while all Ca2+-ATPases declined by approximately 60-80%. This is the first report demonstrating the successful isolation of freshwater chloride cells and also an exclusive method of getting high-purity seawater chloride cells. The isolated cells are viable and suitable for further cytological and molecular studies to elucidate the mechanisms of ionic transport.